Programmed cell death is an essential process by which unwanted or defective cells are removed in an orderly fashion. The engulfment of dying cells, therefore, must be rapid and complete, without releasing the contents of the dying cells. Failures in cell death lead to deformities and cancer. Failure to engulf dying cells may also result in morphogenetic defects, patterning errors, secondary necrosis and inflammation. The study of cell engulfment has primarily focused on the phagocytic cells. Several Drosophila and C. elegans genes required for engulfment have been identified. Surprisingly, none are required exclusively in the dying cell. We have very little understanding of the cellular changes within the dying cell that promote its engulfment. We recently made an important advance to understanding the engulfment of dying cells during Drosophila embryogenesis by developing a sensitive assay for engulfment in living embryos using a fluorogenic, engulfment substrate, which we call VGAL. This engulfment assay showed that the pattern of engulfment faithfully mirrors the pattern of cell death. Surprisingly, the pattern of cell engulfment was unperturbed in embryos that were unable to activate their caspases, indicating that the signal for cell engulfment is independent of the caspase activation cascade. Caspase-independent cell engulfment is a new and potentially important phenomenon that we know very little about. Caspase-independent engulfment challenges the dogma that the caspase cascade controls the engulfment process. This exploratory, R21 grant proposal is intended to create new tools for visualizing and manipulating engulfment in vivo. A significant limitation of VGAL is that it must be injected in order to detect engulfment, which limits its use for genetic screening. The first aim is to develop a GFP-based cell engulfment reporter that will provide a screening method for engulfment defects and further confirm caspase- independent engulfment. This GFP-based reporter can also be used to study autophagy. The second aim will test a novel hypothesis that cytoskeletal changes within dying, epithelial cells permit/promote the engulfment of dying cells by their healthy neighbors. This model attempts to explain why all known engulfment mutations are required in the engulfing cells, but not the dying cells. Successful completion of both aims will have a significant impact on the study of developmentally-regulated cell death and engulfment. [unreadable] [unreadable]